Although both acute and chronic viral infections remain a major health care burden, the therapeutic options are limited. Some of the most concerning of the viral infections include those caused by the Filoviruses and the Arenaviruses. These RNA viruses cause severe hemorrhagic fevers that are often lethal. Early defense against all viruses is mediated by the innate immune system and the responses triggered by pattern recognition receptors that induce the cytokines and chemokines that orchestrate much of the local and systemic anti-viral defenses. However, in addition to these well- defined innate immune signaling pathways, less well-understood cell autonomous factors also contribute to protect the host from and during viral infection. Based on emerging data and our preliminary studies, we hypothesize that an important strategy of host resistance against viruses is the upregulation of `restriction factors' that prevent viral entry, restrict the ability of viruses to infect or replicate in host cells or increase the ability of cells to withstand viral-induced cytopathy. However, although these restriction factors are likely to provide a wealth of new targets for therapeutic intervention, the identities of such cell-autonomous host factors that protect against viruses are largely unknown. Here we propose to utilize and further optimize a novel unbiased screening approach using transposon mutagenesis. This strategy relies three steps: (i) piggyBac transposon mutagenesis to generate a library of mutagenized cells, (ii) selection of mutant clones resistant to viral-induced cell death and (iii) sequencing to identify transposon insertions sites and candidate genes that contribute to protect the mutant cells from the selection agent. In the R21 phase of this proposal we aim to further develop and utilize this approach for identification of antiviral restriction factors. Additionally, in the R33 phase we propose to both validate our new host targets using native virus as well as modify the transposon system for drug discovery.